1. Field of the Invention
The present invention relates to the enzymatic resolution of lactams. The method of the present invention is useful in preparing compounds which may have utility as pharmaceutical, agricultural and veterinary products or starting materials and intermediates for their synthesis.
2. Discussion of the Prior Art
It is known in the art that chiral resolution of compounds can be achieved by using enzymes. Chiral resolution using enzymes such as esterases on aliphatic esters and cyclic compounds containing esters are described in, for example, W. Boland et al., Synthesis, 1049-1072, 1991. Chiral resolutions using enzymes in aliphatic methyl esters hydrolysis is described in, for example, H. Ohta et al., Chem. Lett.,657-660, 1992. Chiral resolutions using enzymes in cyclohexanes systems are described in, inter alia, M. Ohno, Tet. Lett., 29, 6961-6964, 1988; H. Hemmerle, Tet Lett., 28, 3471-3474, 1987; and B. Brion, Tet Lett., 33, 4889-4892, 1992. Chiral resolutions using lipases or Acetylcholine esterases on cycloheptanes containing diacetates is found in A. J. Pearson et al., JOC, 54, 3882, 1989. Beta-lactams have been reported to be selectively acylated by lipase at the nitrogen function (C. Sih et al., JOC, 58, 1068, 1993).
However, there is no prior art for the enzymatic resolution of lactam esters. It is often desired to obtain a single enantiomer of a racemic lactam ester. These compounds can be used as intermediates for preparing compounds which have utility as starting materials and intermediates for the synthesis of pharmaceutical, agricultural and veterinary products. For example, the enanantiomerically pure form of 7-carbomethoxycaprolactam is a useful intermediate in the synthesis of pharmaceutical drug candidates.
The present invention is directed to a method of separating enantiomeric lactam esters. The lactam esters are contacted with a biocatalyst, such as an enzyme or a microorganism, in an aqueous solution, an organic solvent, or a mixture of organic and aqueous solvents, wherein only one enantiomer is selectively hydrolyzed to give the optically active isomer of the corresponding acid. The hydrolysis product is then separated from the unreacted lactam esters using standard methods known to those skilled in the art. This invention also discloses a novel method for the recycling and re-use of the enzymes as well as the racemization of either enantiomer of the lactam ester after enzymatic resolution.
The present invention is directed to contacting racemic esters of lactam with a biocatalyst, such as an enzyme or a microorganism, whereby one of the optical isomers is selectively hydrolyzed to give the optically active isomer of the corresponding acid. The optically active products are then isolated/purified using suitable procedures.
For illustrative purposes only, the process of the present invention is demonstrated by the following example of enzymatic cleavage of a racemic 7-carbomethoxy caprolactam wherein the S configuration is converted to its acid: 
For convenience, the procedure is described herein using racemic esters of lactam; however, the method of the present invention is not limited to use with the racemic form. The lactam ester may be present in the optical active form or in nonracemic mixtures which have an excess of one of the optical isomers. The method of the present invention allows the lactam mixture to react such that only one of two enantiomeric esters of the lactam is converted to its acid.
The term xe2x80x9cstereoselective hydrolysisxe2x80x9d refers to the preferential hydrolysis of one enantiomer relative to another. The term xe2x80x9cmixturexe2x80x9d as used herein in relation to enantiomeric compounds, denotes mixtures having equal (racemic) or nonequal amounts of enantiomers. The term xe2x80x9cresolutionxe2x80x9d denotes partial, as well as, preferably, complete resolution. The term xe2x80x9cenzymatic processxe2x80x9d or xe2x80x9cenzymatic methodxe2x80x9d or xe2x80x9cenzymatic reactionxe2x80x9d denote a process or method or reaction of the present invention employing an enzyme or microorganism. The term xe2x80x9cenantiomeric excess(es)xe2x80x9d is related to the older term xe2x80x9coptical purityxe2x80x9d. In a mixture of a pure enantiomer (R or S) and a racemate, enantiomeric excess is the percent excess of the enantiomer over the racemate. It can be expressed in the following equation, for example:       Optical    ⁢          xe2x80x83        ⁢    purity    =            percent      ⁢              xe2x80x83            ⁢      enantiomeric      ⁢              xe2x80x83            ⁢      excess        =                                                      [              R              ]                        -                          [              S              ]                                                          [              R              ]                        +                          [              S              ]                                      xc3x97        100            =                        %          ⁢          R                -                  %          ⁢          S                    
The enzyme may be any enzyme obtainable from animals, plants, microorganisms, etc. The enzyme may be employed in any conventional form such as in a purified form, a crude form, a mixture with other enzymes, a microbial fermentation broth, a fermentation broth, a microbial body, a filtrate of fermentation broth, and the like, either solely or in combination. In addition, the enzyme or microbial body may be immobilized on a resin.
The activities of the enzymes used in this invention are expressed in xe2x80x9cunitsxe2x80x9d. Units are defined as the rate of hydrolysis of p-nitrophenyl proprionate per minutes as expressed in xcexcmol/min at room temperature.
Specific examples of the enzyme are those obtained from animal and plants such as cow liver esterase, pig liver esterase, pig pancreas esterase, horse liver esterase, dog liver esterase, pig phosphatase, amylase obtainable from barley and potato and lipase obtainable from wheat. Other examples are hydrolases obtained from such microorganisms as Rhodotorula, Trichoderma, Candida, Hansenula, Pseudomonas, Bacillus, Achromobacter, Nocardia, Chromobacterium, Flavobacterium, Rhizopus, Mucor, Aspergillus, Alkaligenes, Pediococcus, Klebsiella, Geotrichum, Lactobaccilus, Cryptococcus, Pichia, Aureobasidium, Actinomucor, Enterobacter, Torulopsis, Corynebacterium, Endomyces, Saccaromyces, Arthrobacter, Metshnikowla, Pleurotus, Streptomyces, Proteus, Gliocladium, Acetobacter, Helminthosporium, Brevibacterium, Escherichia, Citrobacter, Absidia, Micrococcus, Microbacterium, Penicillium and Schizophyllium as well as from lichen and algae.
Specific examples of the microorganisms useful in the present invention include, but are not limited to, Rhodotorula minuta, Rhodotorula rubra, Candida krusei, Candida cylindracea, Candida tropicalis, Candida utilus, Pseudomonas fragi, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Rhizopus chinensis, Mucor pusillus, Aspergillus niger, Alkaligenes faecalis, Torulopsis ernobii, Bacillus cereus, Bacillus subtilis, Bacillus pulmilus, Bacillus subtilis var. niger, Citrobacter freundii, Micrococcus varians, Micrococcus luteus, Pediococcus acidlactici, Klebsiella pneumoriae, Absidia hyalospora, Geotrichun candidum, Schizophyllum commune, Nocardia uniformis subtsuyanarenus, Nocardia uniformis, Chromobacterium chocolatum, Hansenula anomala var. ciferrii, Hansenula anomala, Hansenula polymorpha, Achromobacter lyticus, Achromobacter parvulus, Achromobacter sinplex, Torulopsis candida, Corynebacterium sepedonicum, Endomyces geotrichum, Saccaromyces carrvisial, Arthrobacter globiformis, Streptomyces grisens, Micrococcus luteus, Enterobacter cloacae, Corynebacterium ezui, Lacto bacillus casei, Cryptococcus albidus, Pichia polimorpha, Penicillium frezuentans, Aureobasidium pullulans, Actinomucor elegans, Streptomyces grisens, Proteus vulgaris, Gliocladium roseum, Gliocladium virens, Acetobacter aurantius, Helminthosporium sp. Chromobacterium iodinum, Chromobacterium violaceum, Flavobacterium lutescens, Metschnikowia pulcherrima, Pleurotus ostreatus, Brevibacterium ammoniagenes, Brevibacterium divaricatum, Escherichia coli, Rodotolura minuta var. texensis, Trichoderma longibrachiatum, Mucor javanicus, Flavobacterium arbonescens, Flavobacterium heparinum, and Flavobacterium capsulatum. 
Exemplary, commercially available enzymes suitable for use in the present invention include lipases such as Amano PS-30 (Pseudomonas cepacla), Amano GC-20 (Geotrichum candidum), Amano APF (Aspergillus niger), Amano AK (Pseudomonas sp.), Pseudomonas fluorescens lipase (Biocatalyst Ltd.), Amano Lipase P30 (Pseudomonas sp.), Amano P (Pseudomonas fluorescens), Amano AY-30 (Candida cylindracea), Amano N (Rhizopus niveus), Amano R (Penicillium sp.), Amano FAP (Rhizopus oryzae), Amano AP-12 (Aspergillus nlger), Amano MAP (Mucor melhei), Amano GC-4 (Geotrichum candidum), Sigma L-0382 and L-3126 (porcine pancrease), Lipase OF (Sepracor), Esterase 30,000 (Gist-Brocarde), KID Lipase (Gist-Brocarde), Lipase R (Rhizopus sp., Amano), Sigma L-3001 (Wheat germ), Sigma L-1754 (Candida cytindracea), Sigma L-0763 (Chromobacterlum vlscosum) and Amano K-30 (Aspergillus nlger). Additionally, exemplary enzymes derived from animal tissue include esterase from pig liver, chymotrypsin and pancreatin from pancreas such as Porcine Pancreatic Lipase (Sigma). Two or more, as well as a single, enzyme may be employed when carrying out the process of the present invention.
In addition, enzymes which are serine carboxypeptidases can be used. These enzymes are derived from Candida lipolytica, Saccharomyces cerevisiae, wheat (Triticum aestivum) and Penicillium janthinellum. Commercially available cross-linked enzyme crystals may also be used such as from Altus Biologics, Inc.(e.g. ChiroCLEC-CR, ChiroCLEC-PC, ChiroCLEC-EC).
The present invention is also directed to the use of thermostable esterases and genetically engineered esterases for the resolution of the lactam esters. These enzymes, commercially available from ThermoGen, Inc., are especially suitable for use in industrial processes and are easy to use. In addition to functioning at a wide range of temperatures including higher temperatures, these thermostable enzymes possess an increased shelf life which improves handling. The enzymes are also able to endure harsh, non-biological conditions (pH, salt concentrations, etc.) usually associated with industrial processes because of their stability under operational conditions. They can be immobilized for reuse in multiple applications and hence improving the cost-effectiveness of the process.
During the isolation of the products after enzymatic resolution, the enzymes are frequently exposed to traces of organic solvents. In addition, some enzymatic resolutions are found to work best under a mixture of aqueous and organic solvents or in organic solvents alone. The esterase enzymes of the present invention are more tolerant to denaturing by many organic solvents compared to conventional enzymes which allows longer operational half lives. Most of the esterase enzymes are produced using genetic engineering techniques of gene cloning which ensures the purity of these enzymes and the ease of process controls during scale up.
It was discovered that many esterase and lipase enzymes offer a high degree of stereoselectivity in the resolution of the lactam esters. The preferred enzymes for the resolutions of lactam esters include the thermoesterases THERMOCAT E002, THERMOCAT E010, THERMOCAT E015, THERMOCAT E020 from ThermoGen, Inc. with the most preferred enzymes being the THERMOCAT E020.
Instead of isolated enzymes, there may also be employed a microorganism which can produce any enzyme as stated above.
The enzyme or microorganisms may be used alone or in combination. Depending upon the type of enzyme or microorganism used, either one of the optical isomers of the lactam ester is predominantly hydrolyzed to give the optically active acid. Either one of the optical isomers may be obtained by the selection of a suitable enzyme or microorganism.
The enzymatic hydrolysis of the present invention may be carried out by contacting the lactam esters with the enzyme or microorganism, usually in an aqueous buffer medium with good agitation.
The buffer medium may be inorganic acid salt buffers (e.g. potassium dihydrogen phosphate, sodium dihydrogen phosphate), organic acid salt buffers (e.g. sodium citrate), or any other suitable buffer. The concentration of the buffer may vary from 0.005 to 2 M, preferably from 0.005 to 0.5 M and will depend on the specific lactam ester and the enzymes microorganism used.
Depending on the solubility of the lactam esters, a surfactant may be added to the reaction mixture to solubilize the substrate; preferred surfactants include but are not limited to nonionic surfactants such as alkylaryl polyether alcohols. A preferred surfactant is octylphenoxy polyethoxyethanol, commercially available as Triton X-100 (from Sigma Chemical Company). An effective amount of a surfactant is used. Typical amounts can vary from 0.05% to about 10%.
It is sometimes preferable to add an effective amount of an organic cosolvent to increase product solubility to facilitate the reaction. Examples of solvents include but are not limited to acetonitrile, THF, DMSO, DMF, alcohols, etc. Effective amounts of a co-solvent includes from 1% to 30% depending on the specific lactam ester and enzymes and/or microorganism used.
The pH of the buffers or the pH of the reaction is normally from 4 to 10, preferably from 5 to 9, most preferably from 7 to 8. The reaction temperature may vary from 0 to 100xc2x0 C. and will depend on the specific lactam ester and the enzymes or microorganism used. The reaction time is generally from 1 hour to 70 hours and will depend on the specific lactam ester, enzyme concentration and the enzymes the microorganism used. Normally, the enzymatic hydrolysis is allowed to proceed for a period sufficient to generate a satisfactory quantity of the desired esters or acid in satisfactory optical purity. As the reaction progresses, the amount of desired ester or acid and their optical purities may be monitored by HPLC and chiral HPLC. Normally, the conversion is carried to approximately 50%, after which the acid and the esters are usually obtained in good yields after isolation.
The amount of enzyme used could vary widely from 5 units to 12,000 units of enzyme per mole of starting materials. (The activities of the enzymes used in this invention are expressed in xe2x80x9cunitsxe2x80x9d. Units are defined as the rate of hydrolysis of p-nitrophenyl proprionate per minutes as expressed in xcexcmol/min at room temperature). The amount of enzyme needed will depend on the temperature, the specific lactam ester, the enzymes and/or microorganism used, and the desirable reaction time. It may also be desirable to use a large amount of enzymes in some cases to ensure a practically short reaction time, especially when the enzymes are immobilized and can be reused for many turnovers. The concentration of the ester substrate may be from 0.1 g/L to 100 g/L and depends on the specific lactam ester and the enzyme and/or microorganism used.
The enzymes and/or microorganisms used in the present invention may be in crude form or in an immobilized form. They can be immobilized on various solid supports without loss of stereospecificity or change in stereo selectivity. The solid supports can be inert absorbents to which the enzyme is not covalently bonded. Instead the enzyme is absorbed such as by interactions of hydrophobic or hydrophilic portions of a protein with like regions of the inert absorbent, by hydrogen bonding, by salt bridge formation, or by electrostatic interactions. Inert absorbent materials include, but are not limited to, synthetic polymers (e.g. polystyrene, poly-(vinylalcohol), polyethylene and polyamides), mineralaceous compounds (e.g. diatomaceous earth and Fuller""s earth), or naturally occurring polymers (e.g. cellulose). Specific examples of such materials include Celite 545 diatomaceous earth, Abelite XAD-8 polymeric resin beads and polyethylene glycol 8000.
The enzyme may also be immobilized on the support to which the enzyme is covalently bonded (e.g., oxirane-acrylic beads and glutaraldehyde activated supports). Specific examples include Eupergit C oxirane-acrylic beads and glutaraldehyde activated Celite 545. Other possible immobilizing systems are well known and are readily available to those skilled in the art of enzyme immobilization.
Instead of conventional immobilization method described above, it was discovered that the enzymes could also be conveniently recycled for reuse by simply precipitating out the used enzymes with ammonium sulfate. The precipitated enzyme-ammonium sulfate could be used directly in the next enzymatic hydrolysis. Salts are commonly used in purification of enzymes. They generally protect the protein enzymes by reducing solvent activity. It was discovered that ammonium sulfate, potassium sulfate, potassium phosphate, sodium chloride etc. are effective in recovering the enzyme thermoesterase (E020) activity. Among the salts, ammonium sulfate is the most preferred one.
The desired products, the optically pure (or enriched) unreacted ester and the optically pure (or enriched) acid may be isolated from the hydrolysis mixture using conventional methods such as extractions, acid-base extractions, filtration, chromatography, crystallization or combinations thereof. The recovered enzyme or microorganism may be recycled as described above.
In a convenient isolation procedure, after the enzymatic hydrolysis, the pH is adjusted to pH 7.5 to 8 (in the case of immobilized biocatalysts, the biocatalyst is first separated by filtration), the product acid is separated from the unreacted ester by extracting the ester with an organic solvent such as methylene chloride, ethyl acetate, diethyl ether, methyl t-butyl ether, or any other solvent in which the substrate is soluble and stable. Concentration of the organic extracts affords the optically pure (or enriched) unreacted ester. Concentration of the aqueous phase yield the optically pure (or enriched) acid.
The acid can be freed of the buffer salts and enzyme by selective precipitation or chromatography or other methods known to those skilled in the art. These include acidifying the aqueous to pH 3 (or lower) and isolating the acid by extracting the acid with organic solvent such as methylene chloride, ethyl acetate, diethyl ether, methyl t-butyl ether, or any other solvent in which the acid is soluble and stable. Concentration of the organic extracts affords the optically pure (or enriched) unreacted ester and the optically pure (or enriched) acid and which can be purified and freed of the buffer salts and enzyme by selective precipitation or chromatography or other methods known to those skilled in the art.
Either of the optically pure (or enriched) unreacted ester and the optically pure (or enriched) acid could be racemized if so desired. The optically pure (or enriched) unreacted ester could be racemized by heating in the appropriate base under appropriate conditions. Alternatively, the optically pure (or enriched) unreacted ester could be racemized by heating in acid in the presence of an alcohol under appropriate conditions. The optically pure (or enriched) unreacted acid could also be racemized and converted to the racemic esters by heating in acid in the presence of an alcohol under appropriate conditions. In this manner, excellent yields can be achieved of either the optically pure (or enriched) unreacted ester or the optically pure (or enriched) acid by this combination of stereoselective enzymatic hydrolysis and racemization techniques.
In a preferred embodiment, the present invention is directed to the enzymatic 
R1 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, and heteroaryl which may optionally be substituted by one or more of the following alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, haloalkyl;
A is selected from the group consisting of O, S, and NH or N which may be substituted respectively with one or two independent R1 (the R1s need not be the same);
L is selected from the group consisting of no group or alkylene, alkenylene, alkynylene, and xe2x80x94(CH2)mxe2x80x94Dxe2x80x94(CH2)nxe2x80x94;
D is selected from the group consisting of O, S, SO, SO2, Se, SeO, SeO2, Nxe2x80x94R6;
R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, and heteroaryl which may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, haloalkyl;
m=0 to about 7;
n=1 to about 5;
wherein L may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, S(O)R7, S(O)2R7, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: alkyl, amino, alkylamino, dialkylamino, aminoalkyl, and
R7 is alkyl, or aryl;
X is selected from the group consisting of NH, O, S, Se, (CH2)p, and CHxe2x95x90CH;
p=0 to about 4;
Y is selected from the group consisting of NH, O, S, SO, SO2, Se, SeO, SeO2, (CH2)q, CHxe2x95x90CH;
q=0 to about 2;
Z is selected from the group consisting of (CH2)v, CHxe2x95x90CH;
v=0 to about 2;
R2, R3, and R4 are independently selected from alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, S(O)R7, S(O)2R7, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: halogen, alkyl, amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, alkoxy, and
R5 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, S(O)R7, S(O)2R7, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: halogen, alkyl, amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, alkoxy;
R2, R3, may optionally be taken together to form an alicyclic hydrocarbon, heterocyclyl, heteroaryl or aromatic hydrocarbon and said optionally formed ring may be optionally substituted with one or more of alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, S(O)R7, S(O)2R7, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, or haloalkyl.
Preferably, the present invention is directed to the enzymatic resolution of lactams 
R1 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, which may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, and halogen;
L is selected from the group consisting of no group or alkylene, alkenylene, and alkynylene;
wherein L may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, arylamino, aminoaryl, alkylaminoaryl, cyano, and haloalkyl;
X is selected from the group consisting of (CH2)p, and CHxe2x95x90CH;
p=0 to about 4;
Y is selected from the group consisting of NH, O, (CH2)q and CHxe2x95x90CH;
q=0 to about 2;
Z is selected from the group consisting of (CH2)v and CHxe2x95x90CH;
v=0 to about 2;
R2, R3, and R4 are independently selected from alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: halogen, alkyl, amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, and alkoxy;
More preferably the present invention is directed to the enzymatic resolution of 
R1 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, which may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, and halogen;
Y is selected from the group consisting of NH, O, (CH2)q and CHxe2x95x90CH;
q=0 to about 2;
Z is selected from the group consisting of (CH2)v and CHxe2x95x90CH;
v=0 to about 2;
R2, R3, and R4 are independently selected from alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: halogen, alkyl, amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, and alkoxy.
Even more preferably, the present invention is directed to the enzymatic resolution of lactams of the formula (IV): 
R1 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, which may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, and halogen;
Y is selected from the group consisting of NH, O, (CH2)q and CHxe2x95x90CH;
q=0 to about 2;
R2, R3, and R4 are independently selected from alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: halogen, alkyl, amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, and alkoxy.
Most preferred the present invention is directed to the enzymatic resolution of lactams of the formula (V): 
R1 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, which may optionally be substituted by one or more of the following: alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, and halogen;
R2, R3, and R4 are independently selected from alkyl, alkenyl, alkynyl, hydroxy, alkoxy, thiol, thioalkoxy, halogen, nitro, amino, alkylamino, dialkylamino, aminoalkyl, dialkylaminoalkyl, cyano, and haloalkyl, wherein all said substitutions may be optionally substituted with one or more of the following: halogen, alkyl, amino, alkylamino, dialkylamino, aminoalkyl, hydroxy, and alkoxy.
As utilized herein, the term xe2x80x9calkylxe2x80x9d, alone or in combination, means an acyclic alkyl radical containing from 1 to 10, preferably from 1 to 8 carbon atoms and more preferably 1 to 6 carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl and the like.
The term xe2x80x9calkenylxe2x80x9d refers to an unsaturated acyclic hydrocarbon radical in so much as it contains at least one double bond. Such radicals containing from 2 to 10 carbon atoms, preferably from 2 to 8 carbon atoms and more preferably 2 to 6 carbon atoms. Examples of suitable alkenyl radicals include propylenyl, buten-1-yl, isobutenyl, pentenylen-1-yl, 2-2-methylbuten-1-yl, 3-methylbuten-1-yl, hexen-1-yl, hepten-1-yl, and octen-1-yl, and the like.
The term xe2x80x9calkynylxe2x80x9d refers to an unsaturated acyclic hydrocarbon radical in so much as it contains one or more triple bonds, such radicals containing 2 to 10 carbon atoms, preferably having from 2 to 8 carbon atoms and more preferably having 2 to 6 carbon atoms. Examples of suitable alkynyl radicals include ethynyl, propynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, pentyn-2-yl, 3-methylbutyn-1-yl, hexyn-1-yl, hexyn-2-yl, hexyn-3-yl, 3,3-dimethylbutyn-1-yl radicals and the like.
The term xe2x80x9cheterocyclylxe2x80x9d means an unsaturated cyclic hydrocarbon radical with 3 to about 6 carbon atoms, wherein 1 to about 4 carbon atoms are replaced by nitrogen, oxygen or sulfur. The xe2x80x9cheterocyclylxe2x80x9d may be fused to an aromatic hydrocarbon radical. Suitable examples include pyrrolyl, pyridinyl, pyrazolyl, triazolyl, pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl, imidazolyl, indolyl, thiophenyl, furanyl, tetrazolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, 1,3-dioxolanyl, 2-imidazolinyl, imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4H-pyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl, benzo(b)thiophenyl, benzimidazonyl, quinolinyl, and the like.
The term xe2x80x9carylxe2x80x9d means an aromatic hydrocarbon radical of 4 to 16 carbon atoms, preferably 6 to about 12 carbon atoms, more preferably 6 to 10 carbon atoms. Examples of suitable aromatic hydrocarbon radicals include phenyl, naphthyl, and the like.
The term xe2x80x9cheteroarylxe2x80x9d means aromatic hydrocarbon radical of 4 to 16 carbon atoms, preferably 6 to about 12 carbon atoms, more preferably 6 to 10 carbon atoms wherein 1 to about 4 carbon atoms are replaced by nitrogen, oxygen or sulfur.
The terms xe2x80x9ccycloalkylxe2x80x9d or xe2x80x9ccycloalkenylxe2x80x9d means an xe2x80x9calicyclic radical in a ring with 3 to 10 carbon atoms, and preferably from 3 to 6 carbon atoms. Examples of suitable alicyclic radicals include cyclopropyl, cyclopropylenyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-cyclohexen-1-ylenyl, cyclohexenyl and the like.
The term xe2x80x9calkoxyxe2x80x9d, alone or in combination, means an alkyl ether radical wherein the term alkyl is as defined above and most preferably containing 1 to 4 carbon atoms. Examples of suitable alkyl ether radicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy and the like.
The term xe2x80x9chalogenxe2x80x9d means fluorine, chlorine, bromine or iodine.
The term xe2x80x9cprodrugxe2x80x9d refers to a compound that is made more active in vivo.
As used herein, reference to xe2x80x9ctreatmentxe2x80x9d of a patient is intended to include prophylaxis.